Plasmodesmata (PD) are membrane-lined channels that span the plant cell wall, linking the cytoplasm between adjacent plant cells. PD form a continuum of cytoplasmic bridges enabling intercellular transport of solutes, signaling molecules, protein, RNA, and ribonucleoprotein complexes. As PD are essential gatekeepers for cell-to-cell transport during development and for the spread of plant viruses and gene silencing signals, our studies will impact how PD control complex developmental programs and plant defense. As plants are important to human health, our studies will lead to the design of strategies to increase plant defense to plant viruses that pirate PD channels, thereby increasing the productivity of agriculturally important crops. We recently identified genes interrupted in two mutants, isel and ise2, that cause embryos to increase PD mediated traffic of fluorescently tagged probes. ISE1 and ISE2 encode two different types of RNA helicases. ISE1 localizes to PD, and ISE2 localizes to cytoplasmic granules resembling RNA processing bodies. While ISE2 does not localize to PD, it affects PD function, as PD in ise2 mutants exhibit ultrastructural abnormalities. Our overall goals are to determine how ISE1 and ISE2 helicases affect intercellular transport of RNA during embryogenesis and seedling development. The specific aims are: 1. Do ISE1 and ISE2 exhibit RNA helicase activity in vitro? 2. What are the signal sequences that cause ISE1 to localize to PD, and ISE2 to localize to cytoplasmic granules? 3. What are the size limits of RNA transport in isel and ise2 embryos? 4. Examination of ISE1 and ISE2 function at different developmental times in Arabidopsis. 5. Do ISE1 and ISE2 affect the movement of viral RNAs? 6. Do ISE1 and ISE2 affect the movement or production of gene silencing RNAs? 7. What proteins do ISE1 and ISE2 interact with in the cell? 8. How does ISE2 affect PD structure? We will perform a broad swath of approaches including biochemistry, conditional over- or under-expression of ISE1 or ISE2 during development, monitoring of RNA, viral, and gene silencing signal movement via PD, and microarray analyses to determine candidate genes that are mis-expressed in ise2 that will inform hypotheses for how ISE2 regulates PD architecture. Our lab is uniquely poised to carry out the above experiments that will significantly impact our understanding of these influential channels.